Mutations in 3ß-hydroxysteroid-δ8, δ7-isomerase paradoxically benefit epidermal permeability barrier homeostasis in mice.

Inherited or acquired blockade of distal steps in the cholesterol synthetic pathway results in ichthyosis, due to reduced cholesterol production and/or the accumulation of toxic metabolic precursors, while inhibition of epidermal cholesterol synthesis compromises epidermal permeability barrier homeostasis. We showed here that 3ß-hydroxysteroid-δ8, δ7-isomerase-deficient mice (TD), an analog for CHILD syndrome in humans, exhibited not only lower basal transepidermal water loss rates, but also accelerated permeability barrier recovery despite the lower expression levels of mRNA for epidermal differentiation marker-related proteins and lipid synthetic enzymes. Moreover, TD mice displayed low skin surface pH, paralleled by increased expression levels of mRNA for sodium/hydrogen exchanger 1 (NHE1) and increased antimicrobial peptide expression, compared with wild-type (WT) mice, which may compensate for the decreased differentiation and lipid synthesis. Additionally, in comparison with WT controls, TD mice showed a significant reduction in ear thickness following challenges with either phorbol ester or oxazolone. However, TD mice exhibited growth retardation. Together, these results demonstrate that 3ß-hydroxysteroid-δ8, δ7-isomerase deficiency does not compromise epidermal permeability barrier in mice, suggesting that alterations in epidermal function depend on which step of the cholesterol synthetic pathway is interrupted. But whether these findings in mice could be mirrored in humans remains to be determined.

Protease-Activated Receptor-2 Regulates Neuro-Epidermal Communication in Atopic Dermatitis.

Background: Activation of protease-activated receptor-2 (PAR2) has been implicated in inflammation, pruritus, and skin barrier regulation, all characteristics of atopic dermatitis (AD), as well as Netherton syndrome which has similar characteristics. However, understanding the precise role of PAR2 on neuro-immune communication in AD has been hampered by the lack of appropriate animal models. Methods: We used a recently established mouse model with epidermal overexpression of PAR2 (PAR2OE) and littermate WT mice to study the impact of increased PAR2 expression in epidermal cells on spontaneous and house dust mite (HDM)-induced skin inflammation, itch, and barrier dysfunction in AD, in vivo and ex vivo. Results: PAR2OE newborns displayed no overt abnormalities, but spontaneously developed dry skin, severe pruritus, and eczema. Dermatological, neurophysiological, and immunological analyses revealed the hallmarks of AD-like skin disease. Skin barrier defects were observed before onset of skin lesions. Application of HDM onto PAR2OE mice triggered pruritus and the skin phenotype. PAR2OE mice displayed an increased density of nerve fibers, increased nerve growth factor and endothelin-1 expression levels, alloknesis, enhanced scratching (hyperknesis), and responses of dorsal root ganglion cells to non-histaminergic pruritogens. Conclusion: PAR2 in keratinocytes, activated by exogenous and endogenous proteases, is sufficient to drive barrier dysfunction, inflammation, and pruritus and sensitize skin to the effects of HDM in a mouse model that mimics human AD. PAR2 signaling in keratinocytes appears to be sufficient to drive several levels of neuro-epidermal communication, another feature of human AD.

Topical hesperidin prevents glucocorticoid-induced abnormalities in epidermal barrier function in murine skin.

Systemic and topical glucocorticoids (GC) can cause significant adverse effects not only on the dermis, but also on epidermal structure and function. In epidermis, a striking GC-induced alteration in permeability barrier function occurs that can be attributed to an inhibition of epidermal mitogenesis, differentiation and lipid production. As prior studies in normal hairless mice demonstrated that topical applications of a flavonoid ingredient found in citrus, hesperidin, improve epidermal barrier function by stimulating epidermal proliferation and differentiation, we assessed here whether its topical applications could prevent GC-induced changes in epidermal function in murine skin and the basis for such effects. When hairless mice were co-treated topically with GC and 2% hesperidin twice-daily for 9 days, hesperidin co-applications prevented the expected GC-induced impairments of epidermal permeability barrier homoeostasis and stratum corneum (SC) acidification. These preventive effects could be attributed to a significant increase in filaggrin expression, enhanced epidermal ß-glucocerebrosidase activity and accelerated lamellar bilayer maturation, the last two likely attributable to a hesperidin-induced reduction in stratum corneum pH. Furthermore, co-applications of hesperidin with GC largely prevented the expected GC-induced inhibition of epidermal proliferation. Finally, topical hesperidin increased epidermal glutathione reductase mRNA expression, which could counteract multiple functional negative effects of GC on epidermis. Together, these results show that topical hesperidin prevents GC-induced epidermal side effects by divergent mechanisms.

Tight junction properties change during epidermis development.

In terrestrial animals, the epidermal barrier transitions from covering an organism suspended in a liquid environment in utero, to protecting a terrestrial animal postnatally from air and environmental exposure. Tight junctions (TJ) are essential for establishing the epidermal permeability barrier during embryonic development and modulate normal epidermal development and barrier functions postnatally. We now report that TJ function, as well as claudin-1 and occludin expression, change in parallel during late epidermal development. Specifically, TJ block the paracellular movement of Lanthanum (La(3+)) early in rat in vivo prenatal epidermal development, at gestational days 18-19, with concurrent upregulation of claudin-1 and occludin. TJ then become more permeable to ions and water as the fetus approaches parturition, concomitant with development of the lipid epidermal permeability barrier, at days 20-21. This sequence is recapitulated in cultured human epidermal equivalents (HEE), as assessed both by ultrastructural studies comparing permeation of large and small molecules and by the standard electrophysiologic parameter of resistance (R), suggesting further that this pattern of development is intrinsic to mammalian epidermal development. These findings demonstrate that the role of TJ changes during epidermal development, and further suggest that the TJ-based and lipid-based epidermal permeability barriers are interdependent.

Regulation of ABCG1 expression in human keratinocytes and murine epidermis.

ABCG1, a member of the ATP binding cassette superfamily, facilitates the efflux of cholesterol from cells to HDL. In this study, we demonstrate that ABCG1 is expressed in cultured human keratinocytes and murine epidermis, and induced during keratinocyte differentiation, with increased levels in the outer epidermis. ABCG1 is regulated by liver X receptor (LXR) and peroxisome proliferator-activated receptor-δ (PPAR-δ) activators, cellular sterol levels, and acute barrier disruption. Both LXR and PPAR-δ activators markedly stimulate ABCG1 expression in a dose- and time-dependent fashion. PPAR-γ activators also increase ABCG1 expression, but to a lesser degree. In contrast, activators of PPAR-α, retinoic acid receptor, retinoid X receptor, and vitamin D receptor do not alter ABCG1 expression. In response to increased intracellular sterol levels, ABCG1 expression increases, whereas inhibition of cholesterol biosynthesis decreases ABCG1 expression. In vivo, ABCG1 is stimulated 3-6 h after acute barrier disruption by either tape stripping or acetone treatment, an increase that can be inhibited by occlusion, suggesting a potential role of ABCG1 in permeability barrier homeostasis. Although Abcg1-null mice display normal epidermal permeability barrier function and gross morphology, abnormal lamellar body (LB) contents and secretion leading to impaired lamellar bilayer formation could be demonstrated by electron microscopy, indicating a potential role of ABCG1 in normal LB formation and secretion.

IL-6 stimulates but is not essential for stratum corneum formation and permeability barrier development during gestation.

The regulation of epidermal ontogenesis is a complex process. Previous studies have shown that cytokines (IL-1, TNFalpha and IL-6) regulate permeability barrier homeostasis in adult mice. Recently, we reported that IL-1 and TNFalpha accelerate stratum corneum (SC) formation and permeability barrier development in foetal rodents. Here, we determined whether IL-6 also regulates SC formation and permeability barrier development during late gestation. Using a rat skin explant model, we demonstrated that IL-6 accelerates permeability barrier formation in a time- and dose-dependent fashion. This acceleration of barrier formation is attributable to (a) accelerated lamellar membrane maturation, (b) formation of a multi-layer SC and (c) enhanced expression of epidermal differentiation markers. When comparing epidermis of IL-6-deficient (knockout mice) and wild-type foetal mice at days 16-18, we could not detect any abnormalities in either SC formation or the expression of differentiation markers in knockout (KO) mice. In parallel, the basal expression levels of IL-6 mRNA in epidermis and IL-6 protein in amniotic fluid were very low, with only a minimal change in IL-6 receptor mRNA levels in epidermis of days 16-22 foetal mice. These low IL-6 levels may account, at least in part, for the absence of epidermal abnormalities in IL-6 KO mice. In conclusion, exogenous IL-6 accelerates epidermal ontogenesis, but it is not essential for normal epidermal maturation.

Actin reorganization is abnormal and cellular ATP is decreased in Hailey-Hailey keratinocytes.

Actin reorganization and the formation of adherens junctions are necessary for normal cell-to-cell adhesion in keratinocytes. Hailey-Hailey disease (HHD) is blistering skin disease, resulting from mutations in the Ca2+ ATPase ATP2C1, which controls Ca2+ concentrations in the cytoplasm and Golgi of human keratinocytes. Because actin reorganization is among the first responses to raised cytoplasmic Ca2+, we examined Ca2+-induced actin reorganization in normal and HHD keratinocytes. Even though HHD keratinocytes display raised baseline cytoplasmic Ca2+, we found that actin reorganization in response to Ca2+ was impaired in HHD keratinocytes. Defects in actin reorganization were linked to a marked decrease in cellular ATP in HHD keratinocytes, which persists, in vivo, in HHD epidermis. Defective actin reorganization was reproduced in normal keratinocytes in which the intracellular ATP concentration had been lowered pharmacologically. ATP concentrations in undifferentiated keratinocytes markedly declined after extracellular Ca2+ was increased, but then recovered to a new baseline that was approximately 150% of the previous baseline. In contrast, ATP concentrations in HHD keratinocytes did not change in response to increased extracellular Ca2+. This report provides new insights into how the ATP2C1-controlled ATP metabolism mediates Ca2+-induced cell-to-cell adhesion in normal keratinocytes. In addition, these findings implicate inadequate ATP stores as an additional cause in the pathogenesis of HHD and suggest novel therapeutic options.